Method of constructing offspring originating from frozen sperm stem cell

ABSTRACT

The present invention provides a method of producing offspring originating in a spermatogonial stem cell of a donor animal which comprises generating spermatozoa in the reproductive organ of a male recipient animal with the use of frozen spermatogonial stem cells originating in the donor animal to give a male individual for reproduction, and then producing animal individual originating in the spermatogonial stem cell of the donor using the said individual for reproduction.

TECHNICAL FIELD

The present invention relates to reproductive technology, morespecifically, to a method of producing offspring originating from frozenspermatogonial stem cells.

BACKGROUND ART

Currently, a method that uses cryopreserved sperm has become usable inthe treatment of reproductive dysfunction of vertebrates such as human,breeding of domestic animals, preservation of species and the like.Actually, sperm- or embryo-cryopreservation is used in the linepreservation of laboratory animals including mouse and domestic animalssuch as cattle. Generally, intended offspring are obtained in thefollowing manner. First, sperm is freezed and preserved in liquidnitrogen and, after thawing, subjected to artificial insemination or invitro fertilization. The resulting ovum is then transplanted intooviduct of pseudopregnant surrogate parent and an animal having thetransplanted ovum is housed so as to obtain the objective offspring.However, preservation method for sperm differs depending on the species,and a commonly usable method has not been established yet. For example,there are no effective freezing method for sperm of C57BL/6(B6) mousethat is used in experiments. In addition, the fact that nitrogen supplyis necessary for sperm cryopreservation not only makes the long-termpreservation expensive but also requires one to pay special attention,for example, at the time of packaging for transportation. Besides, agiven quantity of sperm is needed to attain fertilization, and hence alarge quantity of sperm must be collected and cryopreserved becausecryopreserved sperm cannot be grown in vitro. However, a large quantityof sperm can hardly be collected even from domestic animals from whichsperm can be obtained with relative ease. It is particularly difficultto preserve a large quantity of human sperm in advance for the treatmentof a subject in danger of infertility caused by testicular deficiencydue to chemotherapy or irradiation therapy.

Further, in the case of immature individuals wherein sperm is notgenerated or those wherein spermatogenesis is inhibited for somereasons, germline cannot be preserved. In an attempt to use freeze-driedsperm, mouse sperm thawed with water was revealed to have thefertilizing capacity. However, said method has not been established yetbecause the fertilizing capacity decreases as the preservation termbecomes longer, for example.

Spermatogenesis of animals generally involves a series ofdifferentiation and development wherein germline stem cells (e.g.,spermatogonia) in testes of human or corresponding male reproductiveorgans proliferate with morphological changes. In the case of human etal., spermatogonial stem cells (spermatogonia) develop to spermatocytesthen to spermatids through meiosis, which spermatids undergomorphological changes to give sperm. Hereinafter, the present inventionwill be described taking spermatogonial stem cell as an example ofgermline stem cell.

Spermatogonial stem cells are, similar to other stem cells, not onlystable during freeze-thaw and able to grow in vitro (Japan PatentApplication No. 2003-110821), but also highly resistant to irradiation,temperature, etc. and easy to handle at the occasion of preservation,transportation or the like. Spermatogonial stem cells have advantagesincluding that they can be obtained from individuals lackingspermatogenesis or having a small amount of sperm, and that theyproliferate in vitro to give a sufficient quantity of sperm even whenonly a small amount of sperm is available. Accordingly, if areproduction technology to produce offspring (individual) originating inspermatogonial stem cell is established, it must be highly useful forbreeding domestic animals, preserving rare species, treating human maleinfertility, fertility restoration of a patient in danger of infertilitydue to irradiation or chemotherapy, and the like. As for the freezing ofspermatogonial stem cells, it has been known that cryopreservation canbe carried out in almost the same manner while maintaining thespermatogenic activity across the species including hamster, rat,monkey, human, cattle, pig, and the like.

The transplantation technique for spermatogonial stem cells has beenreported by Brinster et al. (Brinster, R. L. and Zimmermann, J. W.Spermatogenesis following male germ-cell transplantation. Proc. Natl.Acad. Sci. USA (1994) 91, 11298-11302), which technique comprisespreparing cell suspension of spermatogonial stem cells and introducinginto the seminiferous tubules of the testis. The document however issilent about the spermatogenesis from the frozen spermatogonial stemcells. Brinster et al. then confirmed in vivo that frozen spermatogonialstem cells have a spermatogenic activity (Avarbock, M. R., Brinster, C.J. and Brinster, R. L. Reconstitution of spermatogenesis from frozenspermatogonial stem cells. Nat. Med. (1996) 2, 693-696). However, it hasnot been reported that offspring was produced from a frozenspermatogonial stem cell. It was reported that microinsemination withspermatozoa generated by transplantation of cryopreserved testicularpieces into testis gave offspring originating in said spermatozoa(Shinohara, T et al., Birth of offspring following transplantation ofcryopreserved immature testicular pieces and in-vitro microinsemination.Hum. Reprod. (2002) 17, 3039-3045); however, the testicular piecescontained Sertoli cells and the like. In addition, the microinseminationtechnique is expensive and requires specialized technique, and hence isnot commonly applicable. Accoridingly, development of a method forproducing offspring originating in spermatogonial stem cellseconomically in a reproducible fashion has strongly been demanded.

DISCLOSURE OF THE INVENTION

One of the purposes of the present invention is to establish a method ofproducing offspring originating in spermatogonial stem cells (malegermline stem cells) stably.

The present inventors have succeeded in producing offspring originatingin spermatogonial stem cells of a donor animal by generating spermatozoain the testis of a male recipient using the cryopreserved spermatogonialstem cells of the donor animal to obtain an individual for reproduction,and producing offspring with the use of said individual forreproduction, and established the present invention.

Thus, the present invention provides a method of producing offspringoriginating in a spermatogonial stem cell of a donor animal whichcomprises generating spermatozoa in the reproductive organ of a malerecipient animal with the use of frozen spermatogonial stem cellsderived from the donor animal to give a male individual forreproduction, and preparing an animal individual of the donorspermatogonial stem cell origin using the resulting male individual.

As one embodiment, the present invention provides a method of producingoffspring originating in a spermatogonial stem cell of a donor animalwhich comprises transplanting frozen spermatogonial stem cells into thereproductive organ of a male recipient animal, generating spermatozoa inthe recipient testis to obtain a male individual for reproduction,preparing a fertilized ovum with the use of a spermatozoon derived fromthe male individual, and allowing the resulting fertilized ovum todevelop into an animal individual.

For the purposes of the present invention as disclosed in thedescription and claims, the donor and recipient male animals can beeither the vertebrates or invertebrates.

The term “vertebrate” refers to mammals, birds, fishes, amphibians andreptiles. Preferred vertebrate includes a mammal selected from the groupconsisting of human, non-human primate, dog, cat, goat, pig, mouse, rat,sand rat, hamster, rabbit, pachyderm, horse, sheep, pig, cattle andmarine mammal; and a bird selected from the group consisting of domesticduck, goose, turkey, chicken, ostrich, emu, guinea fowl, pigeon andquail, but is not limited thereto.

The term “invertebrate” refers to echinus, lobster, abalone andcrustacean, but is not limited thereto.

For the present invention, it is preferred that the animal individual isvertebrate and the reproductive organ is testis.

The term “cryopreserved spermatogonial (male germline) stem cell” refersto a frozen preparation comprising spermatogonial stem cells havingspermatogenic activity, and generally includes a thawed preparation. Itwould be clear from the context that which form, i.e., frozen or thawedform, the term refers to. In the present specification, the thawed“frozen spermatogonial stem cell” may be specifically referred to as“freeze-thawed spermatogonial stem cell”.

Transplantation of frozen spermatogonial stem cells into a malerecipient animal can be performed by providing a suspension of frozenspermatogonial stem cells to the seminiferous tubule or the rete testis.

The preparation of fertilized ovum using spermatozoa generated in arecipient male animal can be carried out through natural mating of therecipient male animal with a female animal; artificial reproductionwherein spermatozoa are injected into the genital canal of femaleindividual; or in vitro fertilization-embryo transfer whereinspermatozoon of the recipient male animal and an ovum of a female animalare fertilized extracorporeally, and the resulting fertile ovum is grownfor a given period of time ex vivo and implanted into the uterus. Incase of in vitro fertilization, microinsemination can be employed.

In view of the purposes of the present invention, the male donor andrecipient animals can be mature or immature animals. However, it ispreferred that the male recipient is immature to obtain offspringthrough natural mating.

The method of the present invention can be effected using invertebrateanimals as donor and recipient.

Because offspring can be obtained using a frozen spermatogonial stemcell according to the present invention, it is possible to collectspermatogonial stem cells at an appropriate time, cryopreserved,optionally grown in vitro and further cryopreserved, and then subjectedto the production of offspring as needed. Since the spermatogonial stemcells exist even in an individual lacking spermatogenesis,spermatogonial stem cells can be isolated from an immature individualand preserved, and then transplanted into the same or an allergenicindividual to generate sperm, whereby it became possible to produceoffspring originating in the spermatogonial stem cell. In the case of anindividual in danger of infertility due to degradation or impair ofspermatogenic capacity as the result of chemotherapy or exposure toendocrine disrupter, it is also possible to obtain offspring originatingin spermatogonial stem cells that have been collected from theindividual and stored.

As described above, the present invention makes it possible to preservelines of rare species, laboratory animals, domestic animals and thelike, and restore infertility human patients suspected to becomeinfertile due to chemotherapy, irradiation, or the like. In addition, asoffspring originating in frozen spermatogonial stem cell can be obtainedthrough natural mating, the production of offspring is achievableeconomically without any technology or equipments required for themicroinsemination.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the comparison of the stem cell activities of fresh andfreeze-thawed ROSA26 testis cells after transplantation into infertile Wrecipient mice. (a) Macroscopic appearance of recipient testes aftertransplantation of fresh (3×10⁵ cells injected; left) or frozen-thawed(3×10⁴ cells injected; right) ROSA 26 testis cells. (b) Photomicrographof a histological section of a recipient testis 2 months aftertransplantation with freeze-thawed stem cells.

FIG. 2 shows the fertility restoration of infertile W mice aftertransplantation of freeze-thawed testis cells. (a) A macroscopiccomparison of untransplanted mouse (left) and transplanted recipient(right) testes 241 days after transplantation with frozen stem cells(mouse # 1156). (b) Photomicrograph of a histological section of theuntransplanted W recipient testis. (c) Photomicrograph of a histologicalsection of the transplanted recipient testis. (d) Offspring from aninfertile W recipient male (white, # 1156) that was transplanted withfreeze-thawed, cryptorchid testis cells from a Green mouse.

FIG. 3 is a photograph showing the donor testis cell colonization in thebusulfan-treated, adult recipient testis. (a) Macroscopic appearance ofa recipient testis that received immature green mouse testis cells. (b)Photomicrograph of a histological section of the testis shown in (a).(c) Tubules from epididymis of the testis shown in (a).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be hereinafter described taking mice as anexample; however, one of ordinary skilled in the art would easilyunderstand that the present invention can be put into practice usingmammals other than mouse and further vertebrates and invertebrates withthe use of the method described herein or that known in the art.

The method for preparing frozen spermatogonial stem cells and thawingthe same is substantially the same as that used for other stem cells(Avarbock, M. R. et al., Nat. Med. (1996) 2, 693-696). Specifically, thepreparation can be performed by the method described in a text(Jikken-igaku bessatsu (Experimental Medicine, separate volume) “Stemcell clone, research protocool” Yodosha).

When a sample is collected from an adult donor of which testis containsa small amount of germline stem cells (i.e., undifferentiated cells), itis preferred to be concentrated according to a known method(Jikken-igaku bessatsu, ibid.) before freezing. When a sample iscollected from an immature donor of which testis contains a large amountof male germline stem cells, it can be used without concentration. Forexample, in the case of mouse, about 1-week-old immature individual ispreferably used for preparation. Further, spermatogonial stem cells canbe proliferated in vitro before freezing (Japan Patent Application No.2003-110821). in the method of the present invention, frozenspermatogonial stem cells prepared by any method can be used. Forexample, frozen spermatogonial stem cells can be prepared by isolatingthe whole or a part of testis from a donor animal, removing the tunicaalbuginea in a solvent (PBS: Phosphate-Buffered Saline), dissociatingthe cells with collagenase, trypsin and DNase, suspending the testiscells separated into single cells in a cell cryopreservation solution(Cellbanker; DIA-IATRON, Tokyo) containing dimethyl sulfoxide and fetalbovine serum albumin, transferring aliquots of approximately 1 ml of thecell suspension containing 5×10⁶-10⁷ cells to 1.5 ml cryotubes, andsubjecting to freezing at −80° C. for 1 day. The resulting frozenspermatogonial stem cells can be preserved in liquid nitrogen at −196°C.

Frozen spermatogonial stem cells can be grown in vitro and cryopreservedagain during the preservation period, and be preserved almost forever.At the time of use, frozen cells are thawed and suspended in a solventto give a cell suspension in a conventional manner. Any methods can beused for thawing without limitation. For example, thawing is performedin a water-bath at 37° C. using Dulbecco's modified Eagle's medium(DMEM) containing 10% fetal calf serum (DMEM/FCS). Specifically, frozencells are thawed by adding 10 ml of DMEM/FCS into cryotubes floated in awater-bath. After washing by centrifugation, cells are suspended inDMEM/FCS and kept on ice until transplantation.

The transplantation of spermatogonial stem cells with the use of a stemcell suspension into recipient testis can be performed by any methodsknown in the art. For example, transplantation can be performed bydirect injection into seminiferous tubules through microinjection or byinjection into efferent ducts through microinjection thereby allowing toreach rete testis. The latter is preferred. The transplantedspermatogonial stem cells adhere to the tube wall of the recipientseminiferous tubules, and then differentiate and develop intospermatocytes, spermatids and spermatozoa, and finally mature followingtransfer to the epididymis.

A fertile ovum inseminated with a spermatozoon generated in therecipient testis can be obtained through natural mating with a maleanimal, artificial insemination, or in vitro fertilization-embryotransfer in a conventional manner. The in vitro fertilization-embryotransfer can be carried out by microinsemination. Those methods areknown in the art. When a female animal having a fertile ovum (ova) iskept under appropriate conditions for a certain period of time,offspring of the male donor origin can be obtained.

In the above method, the donor and recipient male animals may be thesame or different individual.

The present invention is further illustrated by the following examples,but should not be limited by them in any respect.

EXAMPLES

In the Examples below, the colonization capacity of frozen and freshspermatogonial stem cells prepared from donor mouse testis were compared(quantification, first experiment), and then offspring originating infrozen spermatogonial stem cells were produced (second experiment).

Donor and recipient mice and methods used for the preparation of donorcells (freeze-thawed spermatogonial stem cells), transplantation ofdonor cells, histological evaluation of recipient testis andmicroinsemination employed in the Examples are described below.

(1) Mouse

1) Donor Mouse (First Experiment)

-   ROSA: B6-TgR(ROSA26)26Sor transgenic mouse purchased from Jackson    Laboratory, USA    Characteristics:

ROSA26 mouse expresses the E.coli LacZ transgene in all of thespermatogenic cells in the seminiferous tubules. The transplanted donortestis cells express β-galactosidase, and hence are stained blue (LacZstaining) in the presence of the substrate 5-bromo-4-chloro-3-indolylβ-D-galactosidase (X-gal). Since the donor mouse expresses the LacZtransgene in all of the spermatogenic cells, the spermatogenesisoriginated from the donor stem cells can be detected by LacZ staining(blue) with the substrate X-gal.

2) Donor Mouse (Second Experiment)

GFP (Green): C57BL/6 Tg14(act-EGFP)OsbY01 transgenic mouse line providedby Dr M. Okabe (Osaka University) (Okabe et al., 1997, FEBS Lett. 407,313-319)

Characteristics:

The spermatogonia and spermatocytes of GFP mice express the gene for theenhanced green fluorescent protein (EGFP), the amount of which graduallydecreases after meiosis. Accordingly, the testis cells can be detectedon the basis of fluorescence intensity. Colonies positive for the EGFPtransgene originating in the donor testis cells were observed with afluorescent stereomicroscope (MZ FLIII, Leica) in the examples below. Inthe experiments, male pups (pup, 6-day-old) or adult mice (adult, 6-8weeks of age) positive for the transgenes were used. Donor testis cellswere collected from the testes of 6-day-old Green mouse pups or from thecryptorchid testes of adult Green mice, 2-3 months after the operation.It has been shown that these testes are enriched for stem cells, due tothe absence of differentiated germ cells, donor testis cells obtainedfrom these mice should improve colonization efficiency and facilitatefertility restoration. Cryptorchid testes were produced as previouslydescribed (Shinohara et al., 2000, Dev. Biol. 220, 401-411).

3) Recipient Mouse (First and Second Experiments)

B6 mouse (busulfan-treated or -untreated): C57BL/6 (B6) mouse (6-12weeks old) purchased from the Shizuoka Laboratory Animal Center (SLC,Japan)

Characteristics:

B6 mice (busulfan-treated) were treated with busulfan (44 mg/kgbodyweight) at 6 weeks of age to destroy the endogenous germ cellsresponsive to spermatogenesis, and used in transplantation 1 month afterbusulfan injection. Busulfan treatment allows donor cell colonization bydestroying the endogenous spermatogonial stem cells. Thus, it mimics theside effects of chemotherapy that occurs in oncology patients.

W mouse: WBB6F1-W/Wv mouse (5-10 days old or 6-12 weeks old, Japan SLC)

Characteristics:

W mice lack endogenous spermatogenesis due to mutations in the c-kittyrosine kinase gene which is normally expressed on germ cells.

Both the busulfan-treated B6 mice and the congenitally infertile W micehave been shown to be capable of generating spermatogenesis fromtransplanted fresh stem cells (Ogawa, T. et al. (2000) Nat. Med. 6,29-34; Shinohara, T. et al., Dev. Biol. 220, 401-411).

(2) Donor Cells (Spermatogonial Stem Cells Derived from Donor);

General Procedures

1) The process generally comprises isolating testis from a donor mouse,and removing the tunica albuginea in PBS, and incubating in Hanks'balanced salt solution containing 1 mg/ml collagenase (Type I) and 7mg/ml DNase at 37° C. for 15 minutes while shaking as appropriate todigest the seminiferous tubules. After removing the separated stromalcells by washing (×2) with PBS, the seminiferous tubules were incubatedin PBS containing 0.25% trypsin at 37° C. for 15 minutes while shakingas appropriate to further digest the seminiferous tubules. Trypsin wasthen inactivated by adding PBS, followed by pipetting to yield a cellsuspension. The suspension was filtered through a nylon mesh (20-30 μm)to remove the undigested cell masses, and centrifuged (600×g) for 5minutes to collect the cells.

When a fresh cell preparation was used for transplantation, a cellsuspension was prepared by suspending the resulting cells in DMEM/FCS.

Cryopreservation of cells were performed by suspending the single testiscell obtained above in Cellbanker (DIA-IATRON, Tokyo) containingdimethyl sulfoxide and fetal bovine serum albumin, transfering aliquotsof 1 ml of the cell suspension (cell density: 10⁷/ml) to 1.5 mlcryotubes, and freezing at −80° C. for 1 day. The frozen spermatogonialstem cells were preserved in liquid nitrogen at −196° C. until use.

2) Thawing of Frozen Donor Cells

Thawing was carried out substantially in accordance with the teaching ofthe supplier of Cellbanker. That is, 10 ml of Dulbecco's modifiedEagle's medium (DMEM) containing 10% FCS (DMEM/FCS) was added dropwiseinto cryotubes floated on a water-bath at 37° C. After washing bycentrifugation (600 g×5 minutes), the cells were resuspended in DMEM/FCSand kept on ice until transplantation.

(3) Transplantation of Donor Cells

Transplantation was performed by introducing a donor testis cellsuspension in DMEM/FCS into the seminiferous tubules or the efferentducts of a recipient mouse by microinjection in a conventional manner(see, Jikken-igaku bessatsu (Experimental Medicine, separate volume)“Stem cell clone, research protocol” Yodosha). The adult (mature) micewere anaesthetized with Avertin injection (640 mg/kg) and the cells weretransplanted into the testes of both sides. The pup (immature) mice wereplaced on ice to cause hypothermia-induced anesthesia and the cells weretransplanted into the testis of only one side. This is to avoid decreaseof postoperative survival rate by the long-term hypothermia. Theexperimental design is summarized in Table 1 below. In the experimentswith B6 mice, approximately 10 μl of the donor testis cell suspensionwas introduced into the seminiferous tubules or the efferent ducts bymicroinjection, whereas only 3 μl of the suspension was injected intothe adult W testis as the testis is small. When immature W recipientswere used as recipients, 2 μl of the donor testis cell suspension wasinjected. In each testis, 75-85% of the tubules were filled with thedonor cells.

In the transplantation of the first experiments, frozen donor cells weresuspended in DMEM/FCS at a concentration of 7.5×10⁶ to 3.0×10⁷ cells/ml,whereas the concentration of fresh cells was 10⁸ cells/ml, because therecovery of frozen-thawed cells varied between experiments.

In the second experiments, the frozen donor cells were suspended inDMEM/FCS at a concentration of either 10⁸ cells/ml (B6 or mature W mice)or 3×10⁷ cells/ml.

(4) Histological Analysis

The histological analysis of recipient mouse testes was performed bymacroscopic or microscopic analysis. The microscopic analysis wascarried out in the following manner.

Testis tissue was fixed in 10% neutral buffered formalin, embedded inparaffin wax and then cut into sections at 12 μm intervals. All sectionswere stained with haematoxylin and eosin. Four histological sectionswere taken from each testis. Each slide was viewed at a magnification of×400 for the analysis. In the second experiment, the numbers of tubulecross-sections with evidence of spermatogenesis (defined as the presenceof multiple layers of germ cells in the entire circumference of theseminiferous tubule) or lacking evidence of spermatogenesis wererecorded for three sections from each testis to assess the level ofspermatogenesis in the recipient testis. At least 500 seminiferoustubules were counted, and statistical analyses were performed usingStudent's t-test.

(5) Microinsemination

Microinsemination was performed by injecting donor testis cellsmicroscopically into oocytes collected from superovulated female mice(C57BL/6xDBA/2 F1) as described (Kimura, Y. et al. (1995) Development121, 2397-2405). Embryos that reached the 2-cell stage after 24 hours inculture were transferred to the oviducts of pseudopregnant ICR females.

The experimental design for the first and the second experiments issummarized in Table I. TABLE I Experimental Design for the First and theSecond Experiments. Donor cell Volume concentration injected No. ofEx.^(a) Donor^(b) Recipient^(c) Type of cells (×10⁶ cell/ml) (μl)recipient 1^(st) Ex. Rosa26, W, adult Frozen 7.5-30 3 6 adult, B6,adult, Frozen 7.5-30 10 4 untreated busulfan W, adult Fresh 100 3 6 B6,adult, Fresh 100 10 4 busulfan 2^(nd) Ex. GFP, B6, adult, Frozen 100 1012 adult, busulfan cryptorchid W, adult Frozen 100 3 9 W, pup Frozen 302 7 GFP, W, pup Frozen 30 2 1 pup^(a)First experiments were designed to evaluate effect of freezing onspermatogonial stem cells; second experiments were designed to deriveoffspring from freeze-thawed spermatogonial stem cells.^(b)ROSA A26 adult: 6-8 weeks old; GFP adult: 14-20 weeks old; GFP pup:6 days old.^(c)W adult: 6-12 weeks old; B6 adult: 10-12 weeks old; W pup: 5-10 daysold.

Example 1 Quantification of Spermatogonial Stem Cells (First Experiment)

The quantity of spermatogonial stem cells in the testis cells collectedfrom donor mouse was determined.

(1) Adult ROSA26 was used as the donor. The freeze-thaw of donor testiscells and the preparation of cell suspension thereof were performedaccording to the method described above in “(2) Donor cells(spermatogonial stem cells originated in donor); General procedures”. Asabove-mentioned, spermatozoa originating in spermatogonial stem cells ofROSA26 develop blue color upon LacZ staining with X-gal, which makes itpossible to quantify spermatogonial stem cells in donor testis cellpreparation.

(2) Transplantation of Donor Testis Cells

Preparation of Cell Suspension

A cell suspension of freeze-thawed donor testis cells in DMEM-FCS whichcontains thawed donor testis cells having been cryopreserved for 5months in liquid nitrogen, and a cell suspension of fresh donor testiscells in DMEM/FCS were used. The viability of cells in the freeze-thawedor fresh cell preparation was examined by trypan blue exclusion.

The viability index of freeze-thawed testis cells was, as indicated bytrypan blue exclusion, significantly reduced compared with that of freshcells not underwent freeze-thawing treatment (viability index offreeze-thawed cells: 67.4±5.9%, mean±SEM, n=8; fresh cells: 93.6±1.7%; ,mean±SEM, n=7; P<0.01). The average recovery for the originally frozencell population was 37.6±5.1% (n=8).

In the frozen-thaw preparations, only live donor testis cells werecounted for transplantation.

Recipients

Congenitally infertile W mice (5-10 days old or 6-12 weeks old) andbusulfan-treated B6 mice (6-12 weeks old) were used.

To examine spermatogenic activity of frozen-thawed stem cells, the samenumber of cells were transplanted to respective recipients. The donortestis cells were used as a suspension in DMEM/FCS.

B6 mice were transplanted with 3 μl suspension of freeze-thawed donortestis cells (7.5×10⁶ to 3.0×10⁷ cells/ml) or 3 μl suspension of freshdonor testis cell (1.0×10⁷ cells/ml).

As described in (4) above, 10 μl each of stem cell suspension wasintroduced into the testis of each recipient under anesthesia by Avertininjection (640 mg/kg).

Mice were kept under the normal conditions for rearing for 2 months andunderwent laparotomy. The testis of each mouse was isolated and stainedwith X-gal. Each blue-stained region of the seminiferous tubules(colonies) represented spermatogenesis from transplanted singlespermatogonial stem cell (Nagano, M. et al. (1999) Biol. Reprod. 60,1429-1436). That is, the other testis cells are irrelevant tospermatogenesis and the endogenous recipient germ cells do not stainpositive with X-gal. Therefore, the number of blue colonies representsthe number of stem cells in the transplanted cell population.

The results are shown in Table II and FIG. 1. FIG. 1 shows thecomparison of stem cell (colonization) activities of fresh andfreeze-thawed ROSA 26 testis cells after transplantation into infertileW recipient mice. (a) Macroscopic appearance of W recipient testes aftertransplantation of fresh (3×10⁵ cells injected; left) or frozen-thawed(3×10⁴ cells injected; right) ROSA 26 testis cells. The blue colorationrepresents donor-derived spermatogenesis, which can be seen asgray/black spots in the black-and-white picture. The level of donorcell-derived spermatogenesis from freeze-thawed donor testis cells ishigher than that from fresh donor testis cells as can be seen from FIG.1 which shows that the number of spermatogenesis in testis is one forthe fresh cells (FIG. 1 a, left) while that is 4 for the freeze-thawedcells (FIG. 1 a, right). It is clear that the freeze-thawed donor cellsexhibit increased level of donor cell-derived spermatogenesis despitelower concentration of injected cells. (b) Photomicrograph of ahistological section of a recipient (W) testis 2 months aftertransplantation with freeze-thawed testis cells. The picture shows thenormal appearance and organization of the germ cells. Haematoxylin andeosin staining was conducted. Scale bars: (a)=1 mm; (b)=25 μm. TABLE IIW recipients Busulfan-treated recipients No. of No. of Type of cellstubules No. or tubules No. of transplanted injected colonies injectedcolonies Freeze/thawed 11 62.1 ± 21.7 8 45.9 ± 16.2 Fresh 11 5.3 ± 2.2 89.0 ± 3.2Values are mean±SEM. The results are based on data from two experimentsfor eache set. Approximately 3 μl was injected into W mice testes,whereas 10 μl was injected into busulfan-treated B6 mice testes.

Table II shows that, in the case of W recipient mice, the extent ofcolony generation from freeze-thawed (frozen) donor testis cells was11.7-times that from fresh donor testis cells (62.1 versus 5.3 coloniesper 3×10⁵ donor cells; P<0.05). Likewise, in the case ofbusulfan-treated B6 recipient mice, the number of colonies fromfreeze-thawed donor testis cells was 5.1-times higher than that fromfresh donor testis cells (P<0.01). The results shown in FIG. 1 and TableII, when taken together, demonstrate that freeze-thawed testis cellshave higher spermatogenic activities than fresh donor cells. Theseresults also indicate that the method of the present invention makes itpossible to perform fertilization through natural mating as a largequantity of spermatozoa can be produced stably, and is suited to apractical use.

Example 2 Fertility Restoration of Recipients Transplanted with FrozenSpermatogonial Stem Cells and Production of Offspring Originating in theDonor Stem Cells (Second Experiment)

(1) The above-mentioned transgenic mice GFP (Green) were used as adonor. The freeze-thawing of donor testis cells and preparation of cellsuspension of donor testis cells were performed according to the methoddescribed above in “(2) Donor cells (spermatogonial stem cellsoriginated in donor); General procedures” Spermatogonial stem cells werecollected from the testis of 6-day-old mouse (pup) or from thecryptorchid testes of adult Green mice, 2-3 months after the operation,and frozen. The spermatogonia and spermatocytes of these transgenic miceexpress the gene for the enhanced green fluorescent protein (EGFP), theamount of which gradually decreases after meiosis.

(2) Transplantation of Donor Cells

Donor Cells for Transplantation

A cell suspension used was prepared according to the method describedabove by preserving frozen donor testis cells in liquid nitrogen for 2-3weeks, thawing and suspending in DMEM/FCS.

Transplantation

Transplantation was performed according to the method described above in“(3) Transplantation of donor cells”.

As designed in Table I, a suspension of donor testis cells from thecryptorchid testes of adult GFP mice was introduced into B6 mice (adult,busulfan-treated) and W mice (adult or pup) by microinjection. Asuspension of donor testis cells from pup GFP mice was introduced into Wmice (pup) by microinjection. Following the transplantation of donortestis cells, the recipient mice were kept under normal conditions forthe animal. As a control, untransplanted animals were kept under thesame conditions.

Production of Offspring by Natural Mating

The recipient mice which had been transplanted with donor testis cellsand kept under the conditions above were housed with B6 wild-typefemales under the normal conditions for these animals to cause naturalmating, and the generation of offspring was examined. The adult and pupmice were subjected to natural mating after 2 weeks and 6 weeks later,respectively.

Histological Analysis of Recipient Testes

The recipient mice were kept under the normal conditions and subjectedto laparotomy 213-246 days after the transplantation of donor testiscells. The testes were isolated from respective mice and subjected tothe evaluation of donor cell colonization efficiency. Histologicalsections of testis were obtained in a similar manner to Example 1, andeach slide was viewed at a magnification of ×400 with an uprightmicroscope for the analysis. To assess the level of spermatogenesis inthe host testis, the numbers of tubule cross-sections with evidence ofspermatogenesis (defined as the presence of multiple layers of germcells in the entire circumference of the seminiferous tubule) or lackingevidence of spermatogenesis were recorded for three sections from eachtestis. At least 500 seminiferous tubules were counted. Statisticalanalyses were performed using Student's t-test.

The testis of fertile recipients which sired offspring, infertilerecipients which failed to sire offspring and control animals which weresimply housed without transplantation was weighted.

The results are shown in Tables III, IV and FIG. 2. FIG. 2 shows theresults of experiments for fertility restoration of infertile W mice bythe transplantation of freeze-thawed testis cells. (a) A microphotographof untransplanted (left) and transplanted (right) recipient testes 241days after transplantation with frozen spermatogonial stem cells (mouse#1156). The figure shows that the transplanted recipient testis isconsiderably larger compared to the untransplanted testis. (b) Amicrophotograph of histological section of the untransplanted Wrecipient testis. (c) A microphotograph of histological section of thetransplanted recipient testis. In FIG. 2 (b), tubules are blank (white)showing that no spermatozoa exist. On the contrary, FIG. 2(c) shows thatmany spermatozoa are generated as indicated by the black spots in theentire circumference of tubules. (d) Offspring from a B6 female (black)mated with an infertile W recipient male (white, 1156) undergonetransplantation with freeze-thawed, cryptorchid testis cells from aGreen mouse. Haematoxylin and eosin staining was performed. Scale bars:(a)=1 mm; (b, c)=50 μm.

Table III shows the combined results from two to three separateexperiments for each type of transplant. Values are mean±SEM.

Table IV shows the detailed data of five recipients that restoredfertility. TABLE III Spermatogenesis after transplantation offreeze-thawed testis cells No. of epididymides with No. of Recipient %Tubules with spermatozoa fertile Type Age No. spermatogenesis^(a) (%)recipients W pup 8 54.5 ± 8.9 7 (87.5) 4 W adult 9 38.4 ± 6.3 5 (55.6) 1B6 adult 12 N.D^(b) N.D^(b) 0 busulfan^(a)Percentage of tubule cross-sections in the recipient testis withspermatogenesis. Seminiferous tubule cross-sections with multiple layersof germ cells were considered to be positive for spermatogenesis.^(b)N.D. = not determined (due to the presence of endogenousspermatogenesis).

TABLE IV Progeny from W recipient mice microinjected with freeze-thawedstem cells Donor Testis % Tubule cross- Fertile cell Days weight^(b)sections with Days to first recipient type^(a) Recipient to analysis(mg) spermatogenesis^(c) progeny^(d) 1150R adult pup 241 44.7 62.7 190 L11.3 N.D. 1156R adult pup 241 58.5 87.6 81 L 12.0 N.D. 1860R adult pup228 23.9 87.7 72 L 6.5 N.D. 1868R pup pup 240 35.8 67.0 136 L 11.5 N.D.2711R adult adult 221 33.7 65.9 221 L 32.2 59.2^(a)Donor cell type: Adult, cryptorchid.^(b)Only right testes were injected with cells in the pup recipients,whereas both testes were injected in the adult recipients.^(c)The rate (%) of recipient testis cross-sections withspermatogenesis. Seminiferous tubule cross-sections with multiple layersof germ cells were considered to be positive for spermatogenesis.^(d)Days from transplantation to birth of first progeny sired by thefemale mice.N.D. = not determined, because donor cells were not transplanted.

Table III shows that four of the eight W (pup) recipient mice havingbeen transplanted with the freeze-thawed donor testis cells restoredfertility and sired offspring within 72-190 days of transplantation. Asshown in Table IV, three of the fertile males received adult cryptorchiddonor testis cells, and one of the fertile males received pup donortestis cells. The donor testis cell origin of the offspring wasconfirmed by green fluorescence under ultraviolet (UV) light.

The mean weight of the fertile recipient testes was significantly higher(40.7±7.3 mg; n=4) than that of the infertile recipient testes (22.6±2.2mg; n=4; P<0.05) or that of the untransplanted control testes (10.4±0.8mg; n=7; P<0.01) (FIG. 2 a). Histological analyses also revealed moreextensive donor germ cell colonization of the fertile recipient testes(77.4±4.8%; n=4) than of infertile recipient testes (27.0±2.3%; n=4;P<0.001) (FIG. 2 b and c).

Spermatogenesis in the recipient testes arose exclusively from donorstem cells, as the stem cells in the W recipient could not undergospermatogenesis. However, the restoration of spermatogenesis occurred inall eight immature W recipient testes, and spermatozoa were observed in87.5% (⅞) of epididymis sections, which suggests potential fertility.The four recipients that produced progeny remained fertile up to thetime of analysis; that is, at least 228 days after transplantation (FIG.2 d; Table 3), which indicates that the transplanted stem cellsunderwent continuous division and normal differentiation.

Taken together, these results demonstrate that the transplantation offrozen stem cells from the testes of pups or cryptorchid adults restorednormal fertility to congenitally infertile W recipients.

By contrast, the restoration of fertility was not so efficient in adultrecipients. Although one of nine adult W recipients achieved fertilitythrough natural mating, the offspring was first obtained 221 days aftertransplantation. The recovery of spermatogenesis was observed in all thecases, but the percentage of the epididymides containing spermatozoa wassmaller as compared with the recipient pups (55.6 versus 87.5%, TableIII). In addition, none of the busulfan-treated recipients becamefertile after 7 months.

FIG. 3 shows donor testis cell colonization in the busulfan-treated,adult recipient testis. (a) A macroscopic appearance of a recipienttestis that received Green pup testis cells. Green seminiferous tubulesunder UV light indicate donor testis cell colonization, which can beseen as white/gray image in the black-and-white picture. (b) Amicrophotograph of histological section of the testis shown in (a).Spermatogenesis is observed in several tubules. However, as shown in(c), tubules from epididymis of the mouse with testis shown in (a)contains no spermatozoa. Haematoxylin and eosin staining were used.Scale bars: (a)=1 mm; (b, c)=200 μm.

Donor-derived spermatogenesis occurred in the testes of both types ofrecipients (W, B6); however, the level of spermatogenesis wassignificantly lower than that in the pup recipients. As can be seen fromthe picture of epididymis, these recipients have smaller number ofspermatozoa and therefore could not make female mice pregnant throughnatural mating. This is also apparent from the histological analysis ofthe busulfan-treated recipient testes examined 12 months aftertransplantation (FIG. 3 b and c).

Microinsemination

In order to overcome the infertility of the busulfan-treated recipientmice which are infertile in natural mating, an in-vitromicroinsemination technique was employed which is commonly used toderive offspring from infertile humans (Palermo, G. et al., (1992)Lancet 340, 17-18; Kimura, Y. et al., (1995) Development 121,2397-2405). One of the busulfan-treated recipient mice was sacrificed180 days after transplantation with frozen-thawed donor testis cells(pup donor-derived), and colonies positive for the EGFP transgeneoriginating in the donor testis cells were observed with a fluorescentstereomicroscope (MZ FLIII, Leica). Live spermatogenic cells wererecovered by repeatedly pipetting colonized tubules. The germ cells thusrecovered were frozen according the previously reported method andstored until microinsemination. After 3-day-storage, the maturespermatozoa or elongated spermatids were injected into female mice(C57BL/6xDBA/2 F1 (B6D2F1)) oocytes (cytoplasm). The oocytes werecollected from superovulated females. About 80% of oocytes developedinto 2-cell forms within 24 h irrespective of the male germ cells used.All 101 diploid zygotes constructed with 80 spermatozoa and 21 elongatedspermatids were transferred into the oviducts, 68 (67%) of which wereimplanted into the uterus and 31 pups in total were born (31%efficiency). Donor origin was confirmed by fluorescence under UV light.The offspring were proven to be fertile.

As described above, it was demonstrated that offspring originating inspermatogonial stem cells can be obtained through natural mating byallowing mature (adult) or immature (pup) recipients to generatespermatozoa using the spermatogonial stem cells derived from immature ormature individuals. The results also demonstrate that the rate ofsuccess for generating offspring through natural mating tends to becomehigher in case of immature recipients compared to mature recipients.

INDUSTRIAL APPLICABILITY

According to the present invention, it became possible to use frozenspermatogonial stem cells in the reproductive technology for vertebratesin place of frozen sperm, which dissolves various problems associatedwith the method using frozen sperm, for example, complicated proceduresrequired for freezing and preservation of sperm, decrease ofreproductive potential of thawed sperm, difficulty in collectingabundant sperm, and the like. Thus, the present invention opens the wayfor establishing the reproductive technology with improved certainty andstability.

The method of the present invention is useful in not only preservationof lines of domestic or laboratory animals, but also protection of rareanimals and treatment of human suffering from congenital infertility oracquired infertility due to treatment of malignant tumors or the like.

1. A method of producing offspring originating in a spermatogonial stemcell, which comprises generating spermatozoa in the reproductive organof a male recipient animal with the use of frozen spermatogonial stemcells derived from a donor animal to give a male individual forreproduction, and producing an animal individual originating in thedonor spermatogonial stem cell using the said individual forreproduction.
 2. The method of claim 1, wherein the frozenspermatogonial stem cells originating in the donor animal istransplanted into the reproductive organ of a male recipient animal,allowing to generate spermatozoa in the recipient reproductive organ togive a male individual for reproduction, producing a fertilized ovumusing the spermatozoa derived from the said individual, and developingthe resulting fertilized ovum into an animal individual.
 3. The methodof claim 2, wherein the fertilized ovum is obtained by natural mating ofthe male individual for reproduction with a female individual, orartificial insemination or in vitro fertilization-embryo transfer. 4.The method of claim 3, wherein the fertilized ovum is obtained bynatural mating of the male individual for reproduction with a femaleanimal.
 5. The method of claim 1, wherein the male recipient animal isimmature animal.
 6. The method of claim 1, wherein the donor andrecipient animals are vertebrates.
 7. The method of claim 6, wherein thevertebrate is selected from mammals, birds, fishes, amphibians andreptiles.
 8. The method of claim 1, wherein the donor and recipientanimals are invertebrates.
 9. The method of claim 1, wherein the frozenspermatogonial stem cells are introduced into the seminiferous tubule orthe rete testis of the male recipient animal.
 10. The method of claim 9,wherein the frozen spermatogonial stem cells are introduced into therete testis through the seminiferous tubule as a guide of the malerecipient animal.
 11. The method of claim 2, wherein the male recipientanimal is immature animal.
 12. The method of claim 3, wherein the malerecipient animal is immature animal.
 13. The method of claim 4, whereinthe male recipient animal is immature animal.
 14. The method of claim 2,wherein the donor and recipient animals are vertebrates.
 15. The methodof claim 3, wherein the donor and recipient animals are vertebrates. 16.The method of claim 4, wherein the donor and recipient animals arevertebrates.
 17. The method of claim 5, wherein the donor and recipientanimals are vertebrates.
 18. The method of claim 2, wherein the donorand recipient animals are invertebrates.
 19. The method of claim 3,wherein the donor and recipient animals are invertebrates.
 20. Themethod of claim 4, wherein the donor and recipient animals areinvertebrates.